LOCUS       sequence-360427-        6523 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Flp-dependent helper virus for monosynaptic tracing; to be 
            coinjected with pAAV-TREtight-mTagBFP2-B19G (Addgene #100799).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6523)
  AUTHORS   Jin L, Sullivan HA, Zhu M, Lavin TK, Matsuyama M, Fu X, Lea NE, Xu 
            R, Hou Y, Rutigliani L, Pruner M, Babcock KR, Ip JPK, Hu M, Daigle 
            TL, Zeng H, Sur M, Feng G, Wickersham IR
  TITLE     Long-term labeling and imaging of synaptically connected neuronal 
            networks in vivo using double-deletion-mutant rabies viruses.
  JOURNAL   Nat Neurosci. 2024 Jan 11. doi: 10.1038/s41593-023-01545-8.
  PUBMED    38212587
REFERENCE   2  (bases 1 to 6523)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Neurosci. 2024 Jan 11. doi: 10.1038/s41593-023-01545-8."
COMMENT     Sequence Label: pAAV-syn-F14F15S-splitTVA-EGFP-tTAv2
FEATURES             Location/Qualifiers
     source          1..6523
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   66..195
                     /label=AAV2 ITR (alternate)
                     /note="Functional equivalent of wild-type AAV2 ITR"
     promoter        229..676
                     /label=hSyn promoter
                     /note="human synapsin I promoter; confers neuron-specific 
                     expression (Kugler et al., 2003)"
     regulatory      683..692
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(961..1581)
                     /codon_start=1
                     /gene="synthetic gene"
                     /product="tetracycline repressor TetR"
                     /label=TetR
                     /note="TetR binds to the tetracycline operator tetO to 
                     inhibit transcription. This inhibition can be relieved by 
                     adding tetracycline or doxycycline."
                     /translation="MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS"
     CDS             complement(1582..1638)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(join(1648..2358,2359..2361,2362..2364))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(1648..2358,2359..2361,2362..2364))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1690..1711)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     2039..2058
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2298..2319
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(2365..2421)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     misc_feature    3029..3617
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3082..3102)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3500..3511)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3625..3849
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3851..3991
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3851..3980
                     /label=AAV2 ITR
     rep_origin      4066..4521
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4153..4172)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4363..4384
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4538..4557)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4657..4679
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4717..4735)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4803..4907
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4908..4976,4977..5768)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5126..5145)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(5939..6523,1..4)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6428..6447
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
       61 catgtcctgc aggcagctgc gcgctcgctc gctcactgag gccgcccggg cgtcgggcga
      121 cctttggtcg cccggcctca gtgagcgagc gagcgcgcag agagggagtg gccaactcca
      181 tcactagggg ttcctgtcga cgctagactg cagagggccc tgcgtatgag tgcaagtggg
      241 ttttaggacc aggatgaggc ggggtggggg tgcctacctg acgaccgacc ccgacccact
      301 ggacaagcac ccaaccccca ttccccaaat tgcgcatccc ctatcagaga gggggagggg
      361 aaacaggatg cggcgaggcg cgtgcgcact gccagcttca gcaccgcgga cagtgccttc
      421 gcccccgcct ggcggcgcgc gccaccgccg cctcagcact gaaggcgcgc tgacgtcact
      481 cgccggtccc ccgcaaactc cccttcccgg ccaccttggt cgcgtccgcg ccgccgccgg
      541 cccagccgga ccgcaccacg cgaggcgcga gatagggggg cacgggcgcg accatctgcg
      601 ctgcggcgcc ggcgactcag cgctgcctca gtctgcggtg ggcagcggag gagtcgtgtc
      661 gtgcctgaga gcgcagtcag ccgccaccat ggttagaagt tcctattcta tcagaagtat
      721 aggaacttcg cagaatggta gctggattgt agctgctatt agcaatatga aacctcttag
      781 aagttcctat tcttatagga gtataggaac ttctcacagc atatccaggt caaaatcatc
      841 aagtgcatca gcagggagca tgtccaaatc aaagtcatcc agggcatcag caggcagcat
      901 gtccaggtca aaatcatcca aggcatcagc aggcacgtgc tttctctttt tgggcacgtg
      961 agagccagac tcacatttca gctgcttttc caggccacag atgatcagtt ccaggccaaa
     1021 aaggaatgca ggctcagccc cttggtggtc aaacaactca atagcttgcc gcagcagtgg
     1081 tggcatggag tcagtggtgg gggtctccct ctcctccttg gcaacttggt gctcctggtc
     1141 ctccagcaca cagcccaagg tgaagtggcc cacagcagag agggcataca aggcattttc
     1201 cagggagaag ccttgttggc acaggaatgc cagctggttc tccagggtct catactgctt
     1261 ctctgtaggc ctggtgccca agtgaacttt ggcaccatcc cggtgggaga gcaaggcaca
     1321 tctgaagctc ttggcattgt tcctcaggaa gtcctgccag gactcccctt ccagagggca
     1381 gaagtgggtg tggtgcctgt ccaacatctc aatggccagg gcatcaagca gagccctctt
     1441 gttcttcaca tgccagtaca gggtaggctg ctccacaccc agcttctggg ccagcttcct
     1501 ggtggtcaag ccctcaatgc caacttcatt caggagctcc aaggcagagt tgatgacttt
     1561 gctcttgtcc agtctggaca taggtccagg gttctcctcc acgtctccag cctgcttcag
     1621 caggctgaag ttagtagctc cgcttccctt gtacagctcg tccatgccga gagtgatccc
     1681 ggcggcggtc acgaactcca gcaggaccat gtgatcgcgc ttctcgttgg ggtctttgct
     1741 cagggcggac tgggtgctca ggtagtggtt gtcgggcagc agcacggggc cgtcgccgat
     1801 gggggtgttc tgctggtagt ggtcggcgag ctgcacgctg ccgtcctcga tgttgtggcg
     1861 gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat agacgttgtg
     1921 gctgttgtag ttgtactcca gcttgtgccc caggatgttg ccgtcctcct tgaagtcgat
     1981 gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg aacttcacct cggcgcgggt
     2041 cttgtagttg ccgtcgtcct tgaagaagat ggtgcgctcc tggacgtagc cttcgggcat
     2101 ggcggacttg aagaagtcgt gctgcttcat gtggtcgggg tagcggctga agcactgcac
     2161 gccgtaggtc agggtggtca cgagggtggg ccagggcacg ggcagcttgc cggtggtgca
     2221 gatgaacttc agggtcagct tgccgtaggt ggcatcgccc tcgccctcgc cggacacgct
     2281 gaacttgtgg ccgtttacgt cgccgtccag ctcgaccagg atgggcacca ccccggtgaa
     2341 cagctcctcg cccttgctca ccatgggtcc agggttctcc tccacgtctc cagcctgctt
     2401 cagcaggctg aagttagtag ctccggatcc ggagaacaag tctgcctggc tcttgtcagg
     2461 caccagcagc tccttggatg cgctttcaag actgaagatg tcagacctgc tttttgcttt
     2521 ggacttcccc catgcagcga taccacccac agctaccagg cagcacagga gcactgcagt
     2581 gatcagcatc cacatgcggc cgtgattcct ggctggcaga gcacgtccag gagcaggggc
     2641 agtgggagcc tctgtgccgt tgtccgtggg caccgcgggg atcgcgctgg tcccgcagcc
     2701 ccactcgtcc cgcccgtcgt cgcagtcggg gtgtccgtcg cacagccagt cctgcgggta
     2761 acactccccg tgggcaccgg gcggctccga gcagcggaac tgaccggggg ggcaacggga
     2821 caaagaaccg ttaccggacc cgttaccggt cacgttaccg ggcagcagca gcagcagcag
     2881 cgcgggcagc agccgcgcac ctccagaagt tcctatactt ctgatagaat aggaacttct
     2941 tgccttaacc cagaaattat cactgttatt ctttagaatg gtgcaaagag aagttcctat
     3001 actcctataa gaataggaac ttctccggaa tcaacctctg gattacaaaa tttgtgaaag
     3061 attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg ctgctttaat
     3121 gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct tgtataaatc
     3181 ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg gcgtggtgtg
     3241 cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct gtcagctcct
     3301 ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg ccgcctgcct
     3361 tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg tgttgtcggg
     3421 gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc tgcgcgggac
     3481 gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc gcggcctgct
     3541 gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc ggatctccct
     3601 ttgggccgcc tccccgcctg gatcctgtgc cttctagttg ccagccatct gttgtttgcc
     3661 cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
     3721 atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg
     3781 ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg
     3841 gctctatggc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg
     3901 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca
     3961 gtgagcgagc gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg
     4021 catctgtgcg gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg
     4081 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
     4141 ccttagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
     4201 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
     4261 tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga
     4321 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
     4381 ctggaacaac actcaactct atctcgggct attcttttga tttataaggg attttgccga
     4441 tttcggtcta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
     4501 aaatattaac gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat
     4561 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     4621 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     4681 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     4741 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     4801 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     4861 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     4921 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     4981 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     5041 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     5101 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg
     5161 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     5221 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     5281 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     5341 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     5401 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg
     5461 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     5521 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     5581 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     5641 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     5701 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     5761 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     5821 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     5881 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     5941 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     6001 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     6061 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca
     6121 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     6181 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     6241 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     6301 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     6361 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     6421 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     6481 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atg
//
